Angiogenesis, the sprouting of new capillaries from small venules, occurs through local degradation of the basement membrane lining the venules followed by migration, alignment and proliferation of endothelial cells toward the angiogenic chemoattractant. Under normal conditions capillary proliferation is tightly controlled in adult tissues and occurs only during the female menstrual cycle in the follicle and corpus luteum, in the placenta during pregnancy, and as a result of bodily injury, such as during wound and fracture repair. A disruption of the balance between stimulatory and inhibitory influences on angiogenesis contributes to a variety of pathological conditions. Persistent angiogenicity occurs in diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, rheumatoid arthritis, hemangiomas, angiofibromas, psoriasis and atherosclerotic plaques, whereas insufficient capillary growth can result in delayed wound healing, nonhealing fractures, ischemia and fetal growth malformations such as hemifacial microsomia (Folkman et al., Science 235: 442-47 (1987)). Neovascularization is also one of the cardinal features that permit neoplastic progression. Tumor growth is critically dependent on new blood supply, and tumors cannot exceed a few millimeters in diameter in isolated perfused organs where capillary endothelium is degenerated (Folkman et al., Cancer 16: 453 (1963)).
Recent analysis of a variety of human cancers have shown that tumor progression occurs through the sequential deregulation and rearrangement of protooncogenes together with the inactivation of tumor suppressor genes. (Fearon et al, Cell 61: 759-67 (1990)). Whether any of these genetic alterations can trigger the disruption of control of angiogenesis in humans is unclear but at least four plausible scenarios can be envisaged which could result in escape from the factors regulating microvascular quiescence. First, these angiogenic factors may cause increased production of diffusible growth factors and cytokines that may either act directly as angiogenic factors to activate endothelial cells or indirectly through the recruitment of other cells that promotes neovascularization through the secretion of molecules with angiogenic potency. Second, they may cause the synthesis of enzymes that allow angiogenic factors such as bFGF to be released from extracellular matrix storages. Third, they may cause the stimulation of adjacent stromal and capillary endothelial cells to produce enzymes such as stromelysin and collagenase which induce basement membrane and extracellular matrix degradation and therefore, promote angiogenesis by allowing endothelial cell detachment and migration from the parent venules into the perivascular stroma. Finally, they may act to disrupt the local synthesis of physiologic inhibitors of angiogenesis.
Astrocytomas are an attractive model for investigation of the relationship between genes which are mutated during tumor. progression and angiogenesis. They are among the most dramatically neovascularized neoplasms with respect to vasoproliferation, endothelial cell cytology and endothelial cell hyperplasia (Brem et al., J. Natl. Canc. Inst. 48: 347-356 (1972)). The malignant progression of astrocytoma is histopathologically and clinically well characterized (Russell et al., Pathology of Tumors of the nervous system (Edward Arnold, London, 1989)) and this progression toward malignancy is accompanied by a well-documented sequential accumulation of genetic alterations (Cavenee et al., Mutat. Res. 247: 199-202 (1991)). Among these, the earliest known event is mutation of the p53 tumor suppressor gene and elimination of its wild type activity (Nigro et al., Nature 342: 705-708 (1989). Sidransky et al., Nature 355: 846-7 (1992). The p53 gene is the most frequently mutated gene in a diverse range of human tumors (Nigro et al, supra; Sidransky et al., supra; Levine et al., Nature 3561: 453-6 (1991)) and intensive investigations have established its role in checkpoint maintenance during the cell cycle (Lane, Nature 358: 15-16 (1992)), in transactivation of the transcription of other genes (Kern et al., Science 252: 1708-1711 (1991); Weintraub et al., Proc. Natl. Acad. Sci. USA 88: 4570-71 (1991); Ginsberg et al., Proc. Natl. Acad. Sci. USA 88: 9979-9983 (1991); Farmeu et al., Nature 358: 83-6 (1992)) in cell growth control (Baker et al., Science 249: 912-915 (1990); Martinez et al., Genes Dev 5: 151-9 (1991); Diller et al., Mol. Cell Biol. 10: 5772-5781 (1990)), in the response of cells to DNA damage (Kastan et al., Canc. Res. 51: 6304-11 (1991), in control of genomic stability (Yin et al., Cell 70: 937-948 (1992)); Livingstone et al., Cell 70: 923-935 (1992)), and in predisposition to some familial human cancers (Malkin et al., Science 250: 1233-8 (1990); Srivastava et al., Nature 348: 747-749 (1990). Much less attention has been paid to its role in intercellular interactions.
An angiogenesis inhibiting factor produced by glioblastoma cells has now been identified. This factor, abbreviated as "GD-AIF" for convenience, was found following experiments where a wild type p53 coding sequence was placed under the control of a conditionally inducible, tetracycline-regulated, promoter activating system. The GD-AIF is useful in a therapeutic context. Additionally, it provides a method by which the sequence of events leading to mutation of p53 and the onset of tumorigenic transformation can be measured. Since wild type p53 induces or stimulates GD-AIF production and the tested mutated p53 does not, by assaying for GD-AIF and/or by assaying levels of the factor, one may diagnose or study the onset and progression of cancers, such as glioblastoma. Hence GD-AIF may also be considered a diagnostic and prognostic marker for patients believed to be suffering from a cancer, such as glioblastoma.
The invention is described in greater detail in the disclosure which follows.